| Plant height is an important agronomic traits in cereal crops,not only determines the plant architecture but also contributes a lot to improve yield potential and yield stability.Therefore,it is essential to explore alternative dwarf mutant with potential values in breeding to broaden the genetic basis of the dwarf traits and supply valuable resource for elucidating the mechanism of agronomic traits regulation genetically and physiologically.NAU9918 is an excellent variety which confers broad spectrum resistance to wheat powdery mildew.However,in the condition of high yield cultivation,wheat variety NAU9918 was weak in lodging-resistance for its tall plant height.In our study,a dwarf plant(NM9)was screened in the mutants created by NAU9918 seeds treated with ethyl methanesulfonate(EMS).To elucidate the genetic mechanism and effects of Rht_NM9 in the mutant NM9,the morphology of mutant NM9,microscopic observation and the results of genetic analysis,gene mapping,gibberellin(GA)response,allellism test and the effect analysis of Rht_NM9 were showed below:1.Phenotypic characterization of the dwarf mutant NM9The seedling height and total tiller number of NM9 were clearly different from that of NAU9918 started at the sixth leaf stage.And the differences were more significant between the mutant and wile-type at the mature stage.The agronomic traits of mutant and the wild-type were measured at the filling stage,the mature stage and after harvest.The plant height of dwarf mutant,NM9(28.95±3.46 cm),was decreased by 68%compred to its wild-type NAU9918(89.95±2.65 cm).And NM9 had additional effective tiller number,elongated spike and grain,decreased flag leaf area and 1000-grain weight.In addition,NM9 was late-maturing compared to its wild-type.For both the number and length of internodes,two key elements that determine plant height,were deceased in NM9,plant height of mutant were decreased dramatically.By frozen sections and microscopic observation at the flowering period,internodes of NM9 were shorter and slimmer than that of NAU9918,mainly because of the reduction in both parenchyma cell length and width.In addition,the flag leaf sampled at the heading stage were uniformly coated with collodion for microscopic observation,the short and narrow flag leaves of NM9 were attributable to the reduction in the length of both long cells and stomata and reduction in the width of long cells,bulliform cells and stomata.2.Genetic analysis of the mutation gene in NM9The genetic pattern underlying dwarfing mutation in NM9 was investigated using the F1 plants,F2 population and corresponding F2:3 lines derived from a cross between NM9 and its wild-type NAU9918.The plant height of F1 heterozygous plants was intermediate between those of the two homozygous parent plants.Genotypes of the F2 plants were classified into three groups,namely,NAU9918 type,NM9 type and heterozygous type,which were deduced from phenotypic information of the corresponding F2:3 lines.These three genotypes were segregated as 1:2:1,and mean values of plant height for the heterozygous type were significantly greater than those for NM9 type,but significantly lesser than those for NAU9918 type,indicating the dwarf mutation in NM9 was caused by a semi-dominant gene Rht_NM9(reduced height of NM9).3.Genetic map construction and mutation gene Rht_NM9 localizationThe F2 population of "NM9×Sumai 3" was established for genetic mapping of the mutation gene.A total of 1920 SSR markers distributed throughout the wheat genome were chosen for initial polymorphism screening between the two parents NM9 and Sumai 3,and then these markers were used for bulked segregation analysis(BSA)and genetic map construction.Then Rht_NM9 was located on the chromosome arm 2AS.For the fine mapping of Rht_NM9,the 90K wheat SNP array was used to genotype two parents and two DNA bulks in BSA(www.illumina.com).SNPs putatively linked to Rht_NM9 were defined as exhibiting polymorphism between the DNA bulks,with expected genotype phasing with NM9 and Sumai 3.Then,the sequences containing these SNP loci were used for designing primers and linkage analysis.Finally,Rht_NM9 was located between SNP markers SNP34 and SNP41 with a genetic distance of 1.9 cM and 1.8 cM,respectively.In addition,the Rht_NM9 linked markers were anchored to the saturated physical map of the chromosome 2A in common wheat,and Rht_NM9 was physically located to a region of 8.86-Mb between 178.90 Mb and 187.76 Mb.The constration of this genetic map laid a foundation for fine mapping and map based cloning of Rht_NM9 gene.4.Evaluation of the responses of Rht_NM9 to GATo evaluate the response of Rht_NM9 to GA,the mutant NM9 and its wild-type NAU9918 were sprayed with GA at the seedling stage.Both NM9 and NAU9918 were insensitive to GA treatment.Because of NM9 carries Rht-B1b,a GA-insensitive gene.To avoid the impact of Rht-Blb in NM9,four homozygous genotypes involving Rht were screened in the F2 population of "NM9xSumai 3",including Rht8,Rht-Blb+Rht8,Rht_NM9+Rht8 and Rht_NM9+Rht-Blb +Rht8,by PCR identification via specific markers.After GA treatment,elongation was observed in only the genotype of Rht8 but not in the genotypes of Rht_NM9+Rht8 or Rht-B1b+Rht8,indicating that Rht_NM9 was a GA-insensitive gene similar to Rht-B1b.5.Analysis for allelism of Rht_NM9,Rht7,Rht8 and Rht21Among the designated Rht genes,Rht7,Rht8 and Rht21 were located on the homoeologous group 2 chromosomes of wheat.Rht7 and Rht21were located on the short arm of chromosome 2A,and Rht8 was located on the short arm of chromosome 2D.For testing allelism,NM9(Rht_NM9,Rht-B1b,Rht8;28.95±3.46 cm)was crossed with XN0004(Rht21,Rht-Blb,Rht8;59.800±1.69 cm).Plant height of the F1 heterozygous plants(52.30±3.53 cm)was lower than that of the parental plants.And transgressive individuals were detected in "NM9×N0004" F2 population;illustrating Rht_NM9 is not an allele of Rht21.Rht7 is a recessive gene with sensitivity to exogenous GA;however,Rht_NM9 is a semi-dominant gene with no sensitivity to GA,indicating that Rht_NM9 was not the same gene as Rht7.In addition,Rht8 is a recessive gene with sensitivity to exogenous GA.The BLASTn search of sequences of the markers linked to Rht8 and Rht_NM9 in the chromosome 2A and 2D database indicated that Rht8 was not the homoeoallele of Rht_NM9.Rht7 was considered to be a homeoallele of Rht8,so Rht_NM9 could not be an allele of Rht7.Therefore,Rht_NM9 was considered to be a new Rht gene in wheat.6.The effects of Rht_NM9 on agronomic traitsTwo F2 segregating populations and corresponding F2:3 lines,derived from NM9 crossed with NAU9918 or Sumai 3,were used to elucidate the effects of Rht_NM9 on multiple agronomic traits.Compared to the genotypic groups without Rht_NM9,plant height was reduced by more than a half,while the effective tiller number per plant,spike length and grain length were significantly increased in the genotypic groups with Rht_NM9.In addition,Yangmail58(Rht-B1b,Rht8)and Chinese Spring(Tall)were crossed with NM9 and NAU9918,and plant height,effective tiller number per plant,spike length and grain length of the hybrids were measured and compared.When NM9 was used as the female parent,plant height decreased to a greater extent in the hybrid than in the hybrid with NAU9918,while effective tiller number per plant,spike length and grain length increased.All these results illustrated that Rht_NM9 had a negative effect on plant height and positive effects on the increase of effective tiller number per plant,spike length and grain length. |
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